BackgroundThe four intrinsic subtypes of breast cancer, defined by differential expression of 50 genes (PAM50), have been shown to be predictive of risk of recurrence and benefit of hormonal therapy and chemotherapy. Here we describe the development of Prosigna™, a PAM50-based subtype classifier and risk model on the NanoString nCounter Dx Analysis System intended for decentralized testing in clinical laboratories.Methods514 formalin-fixed, paraffin-embedded (FFPE) breast cancer patient samples were used to train prototypical centroids for each of the intrinsic subtypes of breast cancer on the NanoString platform. Hierarchical cluster analysis of gene expression data was used to identify the prototypical centroids defined in previous PAM50 algorithm training exercises. 304 FFPE patient samples from a well annotated clinical cohort in the absence of adjuvant systemic therapy were then used to train a subtype-based risk model (i.e. Prosigna ROR score). 232 samples from a tamoxifen-treated patient cohort were used to verify the prognostic accuracy of the algorithm prior to initiating clinical validation studies.ResultsThe gene expression profiles of each of the four Prosigna subtype centroids were consistent with those previously published using the PCR-based PAM50 method. Similar to previously published classifiers, tumor samples classified as Luminal A by Prosigna had the best prognosis compared to samples classified as one of the three higher-risk tumor subtypes. The Prosigna Risk of Recurrence (ROR) score model was verified to be significantly associated with prognosis as a continuous variable and to add significant information over both commonly available IHC markers and Adjuvant! Online.ConclusionsThe results from the training and verification data sets show that the FDA-cleared and CE marked Prosigna test provides an accurate estimate of the risk of distant recurrence in hormone receptor positive breast cancer and is also capable of identifying a tumor's intrinsic subtype that is consistent with the previously published PCR-based PAM50 assay. Subsequent analytical and clinical validation studies confirm the clinical accuracy and technical precision of the Prosigna PAM50 assay in a decentralized setting.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-015-0129-6) contains supplementary material, which is available to authorized users.

IntroductionTumor infiltrating lymphocytes may indicate an immune response to cancer development, but their significance remains controversial in breast cancer. We conducted this study to assess CD8+ (cytotoxic T) lymphocyte infiltration in a large cohort of invasive early stage breast cancers, and to evaluate its prognostic effect in different breast cancer intrinsic subtypes.MethodsImmunohistochemistry for CD8 staining was performed on tissue microarrays from 3992 breast cancer patients. CD8+ tumor infiltrating lymphocytes were counted as intratumoral when in direct contact with tumor cells, and as stromal in adjacent locations. Kaplan-Meier functions and Cox proportional hazards regression models were applied to examine the associations between tumor infiltrating lymphocytes and breast cancer specific survival.ResultsAmong 3403 cases for which immunohistochemical results were obtained, CD8+ tumor infiltrating lymphocytes were identified in an intratumoral pattern in 32% and stromal pattern in 61% of the cases. In the whole cohort, the presence of intratumoral tumor-infiltrating lymphocytes was significantly correlated with young age, high grade, estrogen receptor negativity, human epidermal growth factor receptor-2 positivity and core basal intrinsic subtype, and was associated with superior breast cancer specific survival. Multivariate analysis indicated that the favorable prognostic effect of CD8+ tumor infiltrating lymphocytes was significant only in the core basal intrinsic subgroup (Hazard ratio, HR = 0.35, 95% CI = 0.23-0.54). No association with improved survival was present in those triple negative breast cancers that lack expression of basal markers (HR = 0.99, 95% CI = 0.48-2.04) nor in the other intrinsic subtypes.ConclusionsCD8+ tumor infiltrating lymphocytes are an independent prognostic factor associated with better patient survival in basal-like breast cancer, but not in non-basal triple negative breast cancers nor in other intrinsic molecular subtypes.

BackgroundNanoString’s Prosigna™ Breast Cancer Prognostic Gene Signature Assay is based on the PAM50 gene expression signature. The test outputs a risk of recurrence (ROR) score, risk category, and intrinsic subtype (Luminal A/B, HER2-enriched, Basal-like). The studies described here were designed to validate the analytical performance of the test on the nCounter Analysis System across multiple laboratories.MethodsAnalytical precision was measured by testing five breast tumor RNA samples across 3 sites. Reproducibility was measured by testing replicate tissue sections from 43 FFPE breast tumor blocks across 3 sites following independent pathology review at each site. The RNA input range was validated by comparing assay results at the extremes of the specified range to the nominal RNA input level. Interference was evaluated by including non-tumor tissue into the test.ResultsThe measured standard deviation (SD) was less than 1 ROR unit within the analytical precision study and the measured total SD was 2.9 ROR units within the reproducibility study. The ROR scores for RNA inputs at the extremes of the range were the same as those at the nominal input level. Assay results were stable in the presence of moderate amounts of surrounding non-tumor tissue (<70% by area).ConclusionsThe analytical performance of NanoString’s Prosigna assay has been validated using FFPE breast tumor specimens across multiple clinical testing laboratories.

BackgroundFrom field harvest to the consumer’s table, fresh citrus fruit spends a considerable amount of time in shipment and storage. During these processes, physiological disorders and pathological diseases are the main causes of fruit loss. Heat treatment (HT) has been widely used to maintain fruit quality during postharvest storage; however, limited molecular information related to this treatment is currently available at a systemic biological level.ResultsMature ‘Kamei’ Satsuma mandarin (Citrus unshiu Marc.) fruits were selected for exploring the disease resistance mechanisms induced by HT during postharvest storage. Proteomic analyses based on two-dimensional gel electrophoresis (2-DE), and metabolomic research based on gas chromatography coupled to mass spectrometry (GC-MS), and liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) were conducted. The results show resistance associated proteins were up-regulated in heat treated pericarp, such as beta-1, 3-glucanase, Class III chitinase, 17.7 kDa heat shock protein and low molecular weight heat-shock protein. Also, redox metabolism enzymes were down-regulated in heat treated pericarp, including isoflavone reductase, oxidoreductase and superoxide dismutase. Primary metabolic profiling revealed organic acids and amino acids were down-regulated in heat treated pericarp; but significant accumulation of metabolites, including tetradecanoic acid, oleic acid, ornithine, 2-keto-d-gluconic acid, succinic acid, turanose, sucrose, galactose, myo-inositol, glucose and fructose were detected. Noticeably, H2O2 content decreased, while, lignin content increased in heat treated pericarp compared to the control, which might increase fruit resistibility in response to external stress. Also, flavonoids, substances which are well-known to be effective in reducing external stress, were up-regulated in heat treated pericarp.ConclusionsThis study provides a broad picture of differential accumulation of proteins and metabolites in postharvest citrus fruit, and gives new insights into HT improved fruit disease resistance during subsequent storage of ‘Kamei’ Satsuma mandarin. Interpretation of the data for the proteins and metabolites revealed reactive oxygen species (ROS) and lignin play important roles in heat treatment induced fruit resistance to pathogens and physiological disorders.